Previously, copy DNA (cDNA) of several flaviviruses and other RNA viruses has been cloned in different low copy bacterial vectors to overcome their intrinsic toxicity (due to the large size and cryptic expression of the viral sequences) (Bredenbeek et al. (2003) J. Gen. Virol. 84, 1261-1268; Durbin, et al. (2006) Hum Vaccin. 2, 255-260; Fan and Bird (2008) J. Virol. Methods. 149, 309-315; Li et al. (2011) PLoS One 6, e18197; Pu et al. (2011) J. Virol. 85, 2927-2941; Rice et al. (1989) New Biol. 1, 285-296) Almazán et al. (2008) Methods Mol Biol. 454, 275-91). The cloned cDNAs have been used as templates for production of infectious recombinant viruses, either by in vitro synthesis and transfection of the RNA genomes (Bredenbeek et al., 2003, cited above), or by incorporating the viral cDNA in an expression cassette, which comprise a promoter such as the CMV-IE (Cytomegalovirus Immediate Early) promoter allowing the transcription of the viral RNA from transfected plasmid DNAs (Enjuanes et al. (2001) J. Biotechnol. 88, 183-204; Hall et al. (2003) Proc. Natl. Acad. Sci. USA. 100, 10460-10464). Such viral expression cassettes directing the expression of attenuated foot-and-mouth disease (Ward et al. (1997) J. Virol. 71, 7442-7447) and Kunjin viruses (Hall et al. (2003) Proc Natl Acad Sci USA. 100, 10460-10464) have been used as experimental DNA vaccines. Although the low copy number vector systems comprising a viral expression cassette can be maintained in the bacterial host cell in a stable manner, they have the important disadvantage that they only allow the purification of infective viral cDNA in amounts that are merely sufficient for small scale experimental use. Therefore, their use as a routine source of infective viral cDNA is impossible, for instance in the production of a life cDNA vaccine.
The production of viral DNA vaccines requires a substantial amplification of cloning vectors to obtain sufficient DNA, but these amplification methods are subject to severe constraints. In order to avoid mutations, vectors comprising viral DNA are propagated under conditions which prevent mutagenic events (recombination, mutations, improving mismatch repair, and the like). Bacterial Artificial Chromosomes (BAC) are known for their stability and can contain inserts up to 500 kb or more.
However the size of such a vector with foreign DNA is a serious burden for bacteria, and its replication requires a substantial metabolic effort. Furthermore, exhaustion of nucleotides can lead to increased mutations. Finally, unwanted expression of foreign DNA (so-called cryptic expression) may occur, which can lead to toxic recombinant proteins. The production of toxic proteins by cryptic transcripts is inherent to flavivirus DNA and can only be solved by lowering the copy number of plasmids. Indeed, the higher the copy number of a vector, the higher the concentration of toxic proteins. As a consequence, bacterial hosts may counterselect for mutants wherein these proteins are not expressed.
Pu et al. (2011) J. Virol. 85, 2927-2941, describes in detail various attempts to solve the intrinsic toxicity of flavivirus cDNA in bacteria. These include the in vivo ligation of plasmids comprising parts of the viral genome, specific hosts, mutants to avoid cryptic expression and also low copy number plasmids.
The use of BACs which occur as a single copy in a bacterium provides thus a solution for these problems.
The low copy number is not a drawback for those applications wherein BAC DNA is subsequently subcloned or amplified to increase the concentration and wherein the introduction of some mutations by these techniques is not critical for the envisaged experiments. However, such amplification methods cannot be applied in the manufacture of DNA vaccines, making BACs a non-preferred vehicle for large scale plasmid preparations for DNA vaccines. Very large scale cultures are required to obtain substantial amounts of BAC.
The use of inducible BAC vectors is known from Wild et al. (2002) Genome Res. 12, 1434-1444 whereby the copy number of the BAC increases from 1 copy per cell to up to 100 copies per cell, or even more. Although this system provides a method to increase the yield of BAC DNA, there is a legitimate concern that the strongly increased activity of the replication system upon induction will increase the mutation frequency. The manufacture of DNA vaccines thus requires a system wherein a high copy number of a vector is obtained, but wherein replication of the vectors occurs without intolerable introduction of mutations.